Yang Chien-Chung, Hsiao Li-Der, Yang Chuen-Mao
Department of Traditional Chinese Medicine, Chang Gung Memorial Hospital at Tao-Yuan, Kwei-San, Tao-Yuan 33302, Taiwan.
School of Traditional Chinese Medicine, College of Medicine, Chang Gung University, Kwei-San, Tao-Yuan 33302, Taiwan.
J Inflamm Res. 2020 Nov 20;13:945-960. doi: 10.2147/JIR.S276925. eCollection 2020.
Neuroinflammation, characterized by the increased expression of inflammatory proteins such as matrix metalloproteinases (MMPs), plays a critical role in neurodegenerative disorders. Lipopolysaccharide (LPS) has been shown to upregulate MMP-9 expression through the activation of various transcription factors, including activator protein 1 (AP-1) and forkhead box protein O1 (FoxO1). The flavonoid 3,5,7-trihydroxy-2-phenyl-4H-1-benzopyran-4-one (galangin) has been demonstrated to possess antioxidant and anti-inflammatory properties in various types of cells. Here, we investigated the mechanisms underlying the inhibitory effect of galangin on LPS-induced MMP-9 expression in rat brain astrocytes (RBA-1 cells).
Pharmacological inhibitors and siRNAs were employed to explore the effects of galangin on LPS-challenged RBA-1 cells. Gelatin zymography, Western blotting, real-time PCR, and a luciferase reporter assay were used to detect MMP-9 activity, protein expression, mRNA levels, and promoter activity, respectively. The protein kinases involved in the LPS-induced MMP-9 expression were determined by Western blot. A chromatin immunoprecipitation (ChIP) assay was employed to evaluate the activity of c-Jun at the promoter.
Galangin treatment attenuated the LPS-mediated induction of MMP-9 protein and mRNA expression, as well as the activity at the promoter. In addition, galangin exerted its inhibitory effects on MMP-9 expression through suppressing the LPS-stimulated activation of proline-rich tyrosine kinase (Pyk2), platelet-derived growth factor receptor beta (PDGFRβ), phosphoinositide 3-kinase (PI3K), protein kinase B (Akt), mammalian target of rapamycin (mTOR), and mitogen-activated protein kinases (MAPKs). Pretreatment with galangin attenuated the LPS-induced phosphorylation of c-Jun and FoxO1. LPS-induced cell migration was also suppressed by galangin pretreatment.
Galangin attenuates the LPS-induced inflammatory responses, including the induction of MMP-9 expression and cell migration, via inhibiting Pyk2/PDGFRβ/PI3K/Akt/mTOR/JNK1/JNK2 and p44/p42 MAPK cascade-dependent AP-1 and FoxO1 activities. These results provide new insights into the mechanisms through which galangin mitigates LPS-induced inflammatory responses, and suggest novel strategies for the management of LPS-related brain diseases.
神经炎症以基质金属蛋白酶（MMPs）等炎症蛋白表达增加为特征，在神经退行性疾病中起关键作用。脂多糖（LPS）已被证明可通过激活包括活化蛋白1（AP-1）和叉头框蛋白O1（FoxO1）在内的多种转录因子来上调MMP-9的表达。黄酮类化合物3,5,7-三羟基-2-苯基-4H-1-苯并吡喃-4-酮（高良姜素）已被证明在各种类型的细胞中具有抗氧化和抗炎特性。在此，我们研究了高良姜素对LPS诱导的大鼠脑星形胶质细胞（RBA-1细胞）中MMP-9表达的抑制作用机制。
使用药理抑制剂和小干扰RNA（siRNAs）来探究高良姜素对LPS刺激的RBA-1细胞的影响。分别使用明胶酶谱法、蛋白质印迹法、实时定量聚合酶链反应（PCR）和荧光素酶报告基因检测法来检测MMP-9活性、蛋白表达、mRNA水平和启动子活性。通过蛋白质印迹法确定参与LPS诱导的MMP-9表达的蛋白激酶。采用染色质免疫沉淀（ChIP）试验评估c-Jun在启动子处的活性。
高良姜素处理减弱了LPS介导的MMP-9蛋白和mRNA表达的诱导，以及启动子处的活性。此外，高良姜素通过抑制LPS刺激的富含脯氨酸的酪氨酸激酶（Pyk2）、血小板衍生生长因子受体β（PDGFRβ）、磷脂酰肌醇3-激酶（PI3K）、蛋白激酶B（Akt）、雷帕霉素靶蛋白（mTOR）和丝裂原活化蛋白激酶（MAPKs）的激活，对MMP-9表达发挥抑制作用。高良姜素预处理减弱了LPS诱导的c-Jun和FoxO1的磷酸化。高良姜素预处理也抑制了LPS诱导的细胞迁移。
高良姜素通过抑制Pyk2/PDGFRβ/PI3K/Akt/mTOR/JNK1/JNK2和p44/p42 MAPK级联反应依赖的AP-1和FoxO1活性，减弱LPS诱导的炎症反应，包括MMP-9表达的诱导和细胞迁移。这些结果为高良姜素减轻LPS诱导的炎症反应的机制提供了新的见解，并为LPS相关脑部疾病的治疗提出了新策略。
